1. Field of the Invention
The present invention relates to compositions of matter comprising plant-operable promoter sequences and regulatory sequences derived therefrom and to uses of such compositions to confer gene expression, especially in developing endosperm cells such as in basal endosperm transfer layer cells of the endosperm.
2. Description of the Related Art
To date plants have been genetically modified for a variety of reasons, including to confer pest resistance, e.g., by expressing antifungal or antibacterial proteins, or improving an agronomic trait, e.g., by modulating fruit ripening, or inducing sterility in a hybrid plant or for the large-scale production of proteins for industrial, pharmaceutical, veterinary and agricultural use. In this respect, advances in biotechnological research have produced an explosion of information in relation to the number of nucleic acids identified which, if appropriately expressed, are useful to produce improved plants, for example, plants resistant to pre-harvest sprouting, plants having an improved nutritional quality, plants having a pharmaceutical quality, plants in which reproductive development is controlled, plants having altered shape or size characteristics, plants capable of rapid regeneration following harvest, or plants having improved resistance to pathogens, amongst others.
However, a problem associated with the genetic improvement of agriculturally-important plants, for example, crops, is the manipulation of gene expression to produce plants which exhibit novel characteristics. In this respect, it is often desirable that a nucleic acid to be expressed in a plant is expressed preferentially, selectively, or specifically, in one or more specific cell types, tissues or organs of the plant, or under specific environmental or developmental conditions, rather than being expressed constitutively.
Moreover, as more genes having desirable agronomic or pharmaceutical value become available, the need for transformed plants with multiple genes will increase exponentially. These multiple exogenous genes must typically be controlled by separate regulatory sequences, to provide appropriate levels and patterns of expression which may not be the same for each structural gene or other transgene to be expressed. For example, some genes may need to be expressed constitutively whereas other genes will need to be expressed at certain developmental stages or locations in the transgenic organism. Accordingly, a variety of regulatory sequences having diverse effects is needed.
By “preferentially” as used throughout the specification and claims is meant that a promoter confers expression on a nucleic acid to which it is operably linked to a greater extent or higher level in one or more specific cell types, tissues or organs of a plant, or under specific environmental or developmental conditions than it does in one or more other cells, tissues or organs or under another condition. However, the term “preferentially” does not limit the expression of the nucleic acid to the one or more specific cell types, tissues or organs of a plant, or under specific environmental or developmental conditions. Rather, the level of expression need only be increased to a higher level, and preferably significantly increased. For example, preferential expression may comprise gene expression in BETL that is at least about 1.5-fold the expression detected in endosperm cells other than the BETL layer or in silk tissue, leaves or roots. In another example, preferential expression may comprise gene expression in BETL that is at least about 2-fold the expression detected in endosperm cells other than the BETL layer or in silk tissue, leaves or roots. In another example, preferential expression may comprise gene expression in BETL that is at least about 3-fold the expression detected in endosperm cells other than the BETL layer or in silk tissue, leaves or roots. In anther example, preferential expression may comprise gene expression in BETL that is at least about 4-fold the expression detected in endosperm cells other than the BETL layer or in silk tissue, leaves or roots. In another example, preferential expression may comprise gene expression in BETL that is at least about 5-fold the expression detected in endosperm cells other than the BETL layer or in silk tissue, leaves or roots. In another example, preferential expression may comprise gene expression in BETL that is at least about 10-fold the expression detected in endosperm cells other than the BETL layer or in silk tissue, leaves or roots.
By “selectively” is meant that a promoter confers expression on a nucleic acid to which it is operably linked to in one or more specific cell types, tissues or organs of a plant, or under specific environmental or developmental conditions.
By “specifically” is meant exclusively.
As used throughout this specification and in the claims that follow, and unless the context requires otherwise, the word “confer” and variations thereof such as “conferring” shall be taken to mean the ability of a promoter or an active fragment or derivative thereof, for example in the context of other factors such as DNA conformation and/or cis-acting DNA sequence(s) and/or trans-acting factor(s) and/or signalling pathway(s) and/or transcript structure and/or transcript processing, to produce expression or a pattern of expression of nucleic acid to which the promoter or active fragment or derivative is operably-connected in response to one or more developmental and/or environmental and/or hormonal and/or other stimuli that would normally elicit the expression or pattern of expression for nucleic acid to which the promoter is operably-connected in its native context.
As used throughout this specification and in the claims that follow, the term “promoter” is to be taken in its broadest context and includes transcriptional regulatory sequences of a classical genomic gene, including a basal promoter regulatory region comprising a TATA box which is required for transcription initiation with or without a CCAAT box sequence, and optional additional regulatory elements (e.g., upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or hormonal and/or environmental stimuli, or in a tissue-specific or cell-type-specific manner. A promoter is usually, but not necessarily, positioned upstream, or 5′, of a structural gene, upon which it confers expression. Furthermore, the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of a plant gene.
As used throughout this specification and in the claims that follow, and unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of elements or integers.
As used throughout this specification and in the claims that follow, the term “active fragment” in the context of a promoter shall be taken to mean a fragment or region or portion of a promoter that retains the ability of the promoter from which it is derived to initiate transcription. Such an active fragment need not necessarily confer expression or a pattern of expression on a nucleic acid to which it is operably connected in the same manner as the promoter from which it is derived. For example, an active fragment of a promoter induces the level of expression of a nucleic acid to a higher or lower degree than a promoter from which it is derived. Alternatively, or in addition, an active fragment of a promoter confers expression in a different cell, tissue or organ, or in fewer tissues or in an additional cell, tissue or organ to that in which a promoter from which it is derived confers expression. Methods for identifying such an active fragment will be apparent to the skilled artisan and/or described herein.
As used throughout this specification and in the claims that follow, the term “derivative” in the context of a promoter shall be taken to mean a promoter derived from a promoter as described according to any example hereof, e.g., a promoter comprising one or more additional regulatory elements, e.g., to increase or reduce or otherwise control expression of a nucleic acid operably connected thereto. The present invention also encompasses a derivative comprising a promoter as described according to any example hereof linked to another promoter, e.g., a bi-directional promoter. In this respect, the other promoter may also be a promoter as described according to any example hereof. The term “derivative” also encompasses a promoter comprising a variation in its sequence relative to a promoter as described according to any example hereof. For example, the sequence of such a derivative may include one or more of the following variations: a deletion, an insertion, a single or multiple point mutation or an alteration at a particular restriction enzyme site, provided that the derivative promoter retains its ability to initiate and/or suppress transcription of a nucleic acid linked thereto.
As used throughout this specification and in the claims that follow, the term “expression” or similar term such as “express” shall be taken to refer de minimis to transcription of a nucleic acid to produce RNA and to optionally encompass such transcription and subsequent translation of transcribed RNA to produce a peptide, polypeptide or protein. This definition is not to be limited to any specific cellular context and includes e.g., such expression obtained using in vitro expression systems or in isolated cells, tissues or organs.
Similarly, a “pattern of expression” refers to one or more of the timing, level, cellular location, sub-cellular location, tissue-selectivity or organ-selectivity of expression as hereinbefore defined, including the relative expression in one cell, tissue or organ compared to another cell, tissue or organ, and including the relative level or relative timing of expression such as at different developmental stages or in response to different environmental or hormonal stimuli.
As used throughout this specification and in the claims that follow, the term “operable” will be understood to mean the ability of a stated integer, to function in a particular context albeit not necessarily only in that stated context.
As used throughout this specification and in the claims that follow, the terms “operably connected” and “in operable connection with” mean the positioning of a promoter of the present invention or active fragment or derivative thereof in spatial relation to another nucleic acid, (e.g., a transgene including a structural gene, open reading frame, reporter gene, or nucleic acid encoding a ribozyme, minizyme, RNAi molecule or other RNA) to thereby confer expression on said other nucleic acid by the promoter, active fragment or derivative. Thus, the relative positioning of the promoter, active fragment or derivative to the other nucleic acid produces a structure that confer a functional expression pattern on the other nucleic acid. A promoter is generally positioned 5′ (upstream) to the nucleic acid, the expression of which it controls. To construct heterologous promoter/nucleic acid combinations (e.g., promoter/transgene and/or promoter/selectable marker gene combinations), it is generally preferred to position the promoter at a distance from the gene transcription start site that is approximately the same as the distance between that promoter and the nucleic acid it controls in its natural setting, i.e., the gene from which the promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of promoter function.
As used throughout this specification and in the claims that follow, the term “native context” in the present context shall be taken to mean a genomic gene in which a promoter naturally occurs in the genome of a plant, i.e., from which the promoter is isolated. The genomic gene in which a promoter is located in nature may be identified and/or subjected to sequence comparison using sequence analysis software available from, for example National Center for Biotechnology Information (NCBI) at the National Library of Medicine at the National Institutes of Health of the Government of the United States of America, Bethesda, Md., 20894, United States of America.
In angiosperms, the seed endosperm forms a nutritive tissue for the embryo. For example, the endosperm of cereals originates with a series of free-nuclear divisions, followed by cellularisation and the subsequent formation of a range of functional cellular domains. This tissue is complex in its structure and development, particularly in cereals. The uptake of assimilates by the growing endosperm is a critical process in seed development. The central area of the endosperm consists of large vacuolated cells that store the reserves of starch and highly-abundant storage proteins.
The Basal Endosperm Transfer Layer (BETL) of the endosperm comprises highly specialized transfer cells that facilitate uptake of solutes from maternal pedicel tissue, and translocate the solutes to the developing endosperm and embryo. There is no symplastic connection between maternal and embryonic tissues, and phloem unloading releases nutrients into an apoplastic space. The uptake of nutrients by the endosperm from the apoplast is facilitated by the basal transfer cells, which possess extensive cell wall ingrowths to increase the membrane surface area and transport capacity (Pate et al., Ann. Rev. Plant Physiol. 23 (1972), 173-196). The absence of a basal endosperm cell transfer layer is correlated with reduced rates of grain filling and eventual abortion of seed in maize (Brink and Cooper, Genetics 32, (1947), 350-368; Charlton et al., Development 121 (1995), 3089-3097).
BETL genes may be expressed during the period of maximum grain filling and storage protein deposition in the endosperm e.g., between about 8 to about 20 days after pollination (DAP) in wheat. To date a limited number of BETL-expressed genes have been identified, and these include genes encoding cysteine-rich proteins that contain extensin-like motifs e.g., SPPPP, proteins related at the amino acid sequence level to plant defensins and proteins related at the amino acid sequence level to Bowman-Birk proteases/alpha-amylase inhibitors.
The ability to express a recombinant nucleic acid in endosperm is desirable for the production of heterologous proteins, e.g., for pharmaceutical or industrial purposes. For example, endosperm has evolved to permit the accumulation of large amounts of storage proteins in a small volume and a stable environment. Moreover, the small size of the endosperm permits recombinant proteins to reach a relatively high concentration in a small biomass, which is beneficial for extraction and downstream processing. Such downstream processing is also simplified as a result of low levels of compounds known to interfere with downstream processing steps, such as phenolics and alkaloids present in tobacco leaves and oxalic acid present in alfalfa. Furthermore, because seed is generally suitable for human and animal consumption, accumulation of proteins in developing seed is an attractive means for producing recombinant proteins for oral delivery to humans or animals, e.g., for production of a foodstuff having a pharmaceutical quality, e.g., an oral vaccine or for production of a foodstuff having an improved nutritional quality.
Accumulation of proteins in the seed of a plant is also particularly useful as the harvesting of seed is already a major feature of crop based agriculture and is relatively easy to implement using existing techniques. The selective expression of proteins in endosperm, as opposed to constitutive expression throughout the plant, has a reduced risk of interfering with vegetative plant growth. Moreover, such limited expression limits contact with non-target organisms, such as microbes in the biosphere and leaf-eating herbivores (Stoger et al., Current Opinion in Biotechnology, 16: 167-173, 2005). There is an ongoing need for regulatory sequences that are capable of conferring expression selectively or specifically in the endosperm e.g., because the majority of sequences isolated to date are leaky or non-selective in so far as they confer expression more generally in vegetative or floral tissues or reproductive organs, mature seeds or embryonic tissues, and/or because they are not operable in different species or confer different patterns of expression across species.
Only a few endosperm promoters are known in the art, and these are mostly derived from a few abundantly-expressed storage protein genes. Moreover, the majority of isolated promoters known in the art confer crown cell expression as opposed to basal endosperm transfer cell expression, and there are few examples of promoters conferring a basal endosperm transfer cell-specific expression pattern. Because of the difficulty in expressing multiple genes in plants from the same promoter, the small number of available promoters makes it difficult to modify or improve plant seeds yield or other seed qualities by gene stacking i.e., the expression of multiple transgenes. For example, competition between cis-acting elements for regulatory DNA binding proteins can reduce promoter efficiency such that expression of multiple transgenes under the control of the same promoter in the same cell may be reduced compared to when different promoters are employed.
It will be apparent to the skilled artisan from the foregoing that the genetic manipulation of seed yield and/or seed quality is beneficial to agriculture and achievable e.g., by expressing genes in the endosperm including crown cells and/or BETL cells. The improved plant seeds provide flow-on benefits, permitting the production of pharmaceuticals for human or veterinary use and/or for improving or altering the nutritional quality of a foodstuff produced from a plant. Accordingly, promoters that confer expression in developing endosperm including crown cells and/or BETL cells are clearly desirable to provide these benefits.
Conventional techniques of molecular biology, recombinant DNA technology are described, for example, in the following texts:    1. Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Second Edition (1989), whole of Vols I, II, and III;    2. DNA Cloning: A Practical Approach, Vols. I and II (D. N. Glover, ed., 1985), IRL Press, Oxford, whole of text;    3. Oligonucleotide Synthesis: A Practical Approach (M. J. Gait, ed., 1984) IRL Press, Oxford, whole of text, and particularly the papers therein by Gait, pp 1-22; Atkinson et al., pp 35-81; Sproat et al., pp 83-115; and Wu et al., pp 135-151;    4. Nucleic Acid Hybridization: A Practical Approach (B. D. Hames & S. J. Higgins, eds., 1985) IRL Press, Oxford, whole of text;    5. Perbal, B., A Practical Guide to Molecular Cloning (1984);